Affinity chromatography plays an important role in the research, development and production of proteins, including monoclonal antibodies (Mabs). Affinity chromatography media generally comprises a solid support having a bound ligand capable of interacting with a target molecule. Affinity chromatography is useful because the ligands deployed on solid supports, such as beads, are typically selective for the target molecule. This selectivity allows for good yield, as well as fast and economical purification of target molecules. For immunoglobulins (e.g., IgG), including monoclonal antibodies, Protein A is a selective affinity ligand which binds most sub-classes, Boyle, M. D. P., and Reis, K. J., 1987, Biotechnology, 5: 697. Protein G is another affinity ligand for IgG. Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques, Academic Press, 1992.
Both Proteins A and G can bind more than one IgG. Once immobilized onto a porous chromatography support such as a resin, membrane or other media, both are useful for purification and commercial production of polyclonal IgG or monoclonal antibodies (Mabs).
Protein A may be isolated in its native form from Staphylococcus aureus. Current commercially available Protein A media products such as ProSep vA-HC and n-Protein A Sepharose Fast Flow, use Protein A derived from S. aureus (native or n-Protein A). Other commercial affinity media products such as ProSep-rA and MabSelect® employ Protein A recombinantly produced in E. Coli (recombinant or r-Protein A). See, e.g., U.S. Pat. No. 5,151,350. Other modified recombinant forms of Protein A have also been described. See. e.g., U.S. Pat. No. 5,084,559; U.S. Pat. No. 6,399,750; US. Patent Publication No. 2005/0143566. Protein A ligands comprising a cysteine residue are also known. U.S. Pat. No. 5,084,559; U.S. Pat. No. 6,399,750. The addition of a cysteine amino acid facilitates ligand coupling to the base matrix or resin. Modifications to the B domain of Protein A have also been-described. US Patent Publication No. 2005/0143566.
There are several parameters that may determine the performance of an affinity chromatography media. Selectivity, effective mass transfer, binding capacity and packed bed permeability all play a role in determining the utility of a media to affect the desired separation. Selectivity of the resin may be driven by the ligand and its properties. Many of the performance characteristics are determined by a combination and interplay of the base matrix properties, type of chemical modification used for ligand attachment and ligand properties.
The base matrix plays a role in determining the pressure-flow characteristics of the media and also determines the “effective pore size” into which the target molecule must diffuse to affect adsorption. For synthetic and natural polymers there is often a relationship between the pore size of the material and the observed rigidity. Typically, the larger the pore size the less rigid a material will become. This is especially true for hydrogels such as agarose. Materials made of unmodified agarose have very poor rigidity where only after chemical modification through crosslinking is a desirable rigidity obtained. U.S. Pat. No. 4,973,683. To attempt to overcome these challenges, further chemical crosslinking has been developed which provides particles with suitable pressure-flow properties. See, e.g., U.S. Pat. No. 6,602,990: GE Healthcare Catalog, 2007. However, these materials are still compressible, which limits operating flowrates, moreover pressures are usually restricted to <2 bar, thus limiting productivity and the operating window available to the end-user. Commercial Protein A affinity media made using agarose or modified agarose include Protein A Sepharose Fast Flow®, r-Protein A Sepharose Fast Flow®, Mabselect® and Mabselect Xtra®. US Patent Publication No. 2006/0134805. Mabselect Xtra® is another version of modified agarose where the pore size, particle size and ligand density have been adjusted for the agarose support. GE Healthcare Catalog, 2007.
Silica based solid supports do not suffer from many of the shortcomings associated with agarose supports. One advantage of using solid supports comprising silica (e.g. glass) or ceramic is that they are inherently incompressible. This allows for the decoupling of the media's pore size from the media's mechanical properties. The media's mechanical properties, e.g. compressibility, largely determine the maximum pressure-flow capabilities. Therefore, silica based solid supports, essentially have pore size and pressure-flow properties that are decoupled whereby one property can be altered without affecting the other. Affinity media based on porous silica, particularly controlled pore glass (CPG), have found commercial utility due to their high capacity and suitable pressure-flow characteristics. McCue. J. et. al., 2003, Journal of Chromatography, 989:139. Previously, materials with a pore size of 1000 Å and 700 Å, and particle size of 56 μm to 100 μm have been described. McCue, Justin et. al. Presentation, 225th ACS National Meeting, New Orleans, La., United States, Mar. 23-27, 2003 (2003). Notably the smaller particle size and pore size had the higher capacity, but the capacity improvement was incremental in light of the reduced pressure flow properties. Thus the need for improved capacity remains particularly given the production capacity of large scale preparative methods for protein therapeutics and other biologics.
Currently, the production of monoclonal antibodies is done through the fermentation of mammalian cells in bioreactors on the scale of 10,000-20,000 liters. After clarification, these volumes must be processed through the first chromatography step, which is typically a Protein A column. As fermentation processes and technology improve, the titer concentration in the unprocessed product continually increases, resulting in titers >1 g/L. These higher titer fermentation batches can result in total product protein amounts >20 Kg. Due to the increase in total protein/batch, there is increased demand on the Protein A column binding capacity. Therefore, a need exists to create chromatography materials that have sufficient capacity to bind and purify these large amounts of product protein. Various embodiments of the invention described herein meet this need and others as well.